Identification and characterization of a protostome homologue of peropsin from a jumping spider

Peropsin, a member of the opsin family, has characteristics of two functionally distinct opsin-groups, that is, amino acid residues conserved among opsins for light-sensing and a retinal-photoisomerase-like molecular property. Although such a bilateral feature of peropsin seems to be important for understanding the diversity of the opsin family, previous studies have been limited to higher deuterostome, vertebrate and amphioxus peropsins. Here, we report a protostome peropsin homologue from a jumping spider. We found a spider opsin that shares amino acid homology and conserved amino acid residues with known peropsins. The spider opsin-based pigment heterologously expressed in cultured cells exhibited photoisomerase-like isomerization characteristics and a bistable nature. Based on the characteristics of both the amino acid homology and its photochemical properties, we concluded that the spider opsin is the first protostome peropsin homologue. These results show that peropsin existed before the deuterostome–protostome split like other members of the opsin family. In addition, the spider peropsin was localized to non-visual cells in the retina, and fluorescence from reduced retinal chromophore was also observed in the region where peropsin was localized. These findings provide the first demonstration that the peropsin can form a photosensitive pigment in vivo and underlie non-visual function.     

Crystal structures of MKK4 kinase domain reveal that substrate peptide binds to an allosteric site and induces an auto-inhibition state

MKK4 activates both JNKs and p38s. We determined the crystal structures of human non-phosphorylated MKK4 kinase domain (npMKK4) complexed with AMP-PNP (npMKK4/AMP) and a ternary complex of npMKK4, AMP-PNP and p38α peptide (npMKK4/AMP/p38). These crystal structures revealed that the p38α peptide-bound npMKK4 at the allosteric site rather than at the putative substrate binding site and induced an auto-inhibition state. While the activation loop of the npMKK4/AMP complex was disordered, in the npMKK4/AMP/p38 complex it configured a long α-helix, which prevented substrate access to the active site and αC-helix movement to the active configuration of MKK4.     

Optimization of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives as arginine vasopressin V2 receptor agonists and discussion of their binding modes

A series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives were optimized to achieve potent agonistic activity, both in vitro and in vivo, for the arginine vasopressin V₂ receptor, resulting in the eventual discovery of compound 1g. Molecular modeling of compound 1g with V₂ receptor was also examined to evaluate the binding mode of this series of compounds.     

Glycosphingolipids are not pivotal receptors for Subtilase cytotoxin in vivo: sensitivity analysis with glycosylation-defective mutant mice

Certain glycosphingolipids play important roles as cellular receptor for bacterial toxins with high specificity and strong affinity. In particular AB(5) toxins exhibit typical modes of cell attachment with B5 and invasion and biological effects in cells with A subunit. Subtilase cytotoxin (SubAB) is the prototype of a recently discovered AB(5) cytotoxin family produced by certain strains of Shiga toxigenic Escherichia coli, and shows highly specific serine protease activity toward endoplasmic reticulum chaperone Bip. Since this toxin bound to a mimic of ganglioside GM2, GM2 has been considered to be possible receptor for SubAB. Using six kinds of glycosylation-defective knockout mice lacking certain group of glycosphingolipids, sensitivity to SubAB in vivo was analyzed. Consequently, all mutant mice died at around 70h after intraperitoneal injection of 10 microg (or 7.5 microg) of SubAB as well as wild type mice. These results indicated none of glycolipids are not pivotal receptor for SubAB in the body.     

Sympatric spawning of Anguilla marmorata and Anguilla japonica in the western North Pacific Ocean

Extensive collections were made of the larvae of the temperate Japanese eel Anguilla japonica and the tropical giant mottled eel Anguilla marmorata in an overlapping area of the North Equatorial Current region of the western North Pacific Ocean. Collections of 189 A. marmorata and > 2500 A. japonica larvae during nine surveys from 1991 to 2007 showed that these two anguillid eels have similar spawning areas just west of the southern West Mariana Ridge. In July to August 2006 and August 2007, morphologically and genetically identified A. marmorata preleptocephali were mainly collected between 14·5–15° N and 142–142·5° E, where A. japonica preleptocephali were also caught in some of the same net tows. Fewer A. marmorata preleptocephali, however, were collected ( n = 31) compared to those of A. japonica ( n = c . 165), and fewer small larvae of A. marmorata were collected per tow than A. japonica ( n = 1–10 and 1–294, respectively), suggesting relatively smaller spawning aggregations of A. marmorata . The distribution of preleptocephali and small larvae was wider in longitude in A. marmorata (131– 143° E) than in A. japonica (137–143° E), while the latitudinal range was almost the same (12–17° N). Although spawning by these two species overlaps both spatially and temporally, the tropical eels of the North Pacific population of A. marmorata probably have a much longer spawning season with fewer spawners, at least in summer, and recruit to a much wider latitudinal range of growth habitats.     

Degradation of estrogens by Rhodococcus zopfii and Rhodococcus equi isolates from activated sludge in wastewater treatment plants

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17beta-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17beta-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17beta-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17beta-estradiol into substances without estrogenic activity.     

Variations in the C3, C3a receptor, and C5 genes affect susceptibility to bronchial asthma

Bronchial asthma (BA) is a common chronic inflammatory disease characterized by hyperresponsive airways, excess mucus production, eosinophil activation, and the production of IgE. The complement system plays an immunoregulatory role at the interface of innate and acquired immunities. Recent studies have provided evidence that C3, C3a receptor, and C5 are linked to airway hyperresponsiveness. To determine whether genetic variations in the genes of the complement system affect susceptibility to BA, we screened single nucleotide polymorphisms (SNPs) in C3, C5, the C3a receptor gene (C3AR1), and the C5a receptor gene (C5R1) and performed association studies in the Japanese population. The results of this SNP case-control study suggested an association between 4896C/T in the C3 gene and atopic childhood BA (P=0.0078) as well as adult BA (P=0.010). When patient data were stratified according to elevated total IgE levels, 4896C/T was more closely associated with adult BA (P=0.0016). A patient-only association study suggested that severity of childhood BA was associated with 1526G/A of the C3AR1 gene (P=0.0057). We identified a high-risk haplotype of the C3 gene for childhood (P=0.0021) and adult BA (P=0.0058) and a low-risk haplotype for adult BA (P=0.00011). We also identified a haplotype of the C5 gene that was protective against childhood BA (P=1.4×10^–6) and adult BA (P=0.00063). These results suggest that the C3 and C5 pathways of the complement system play important roles in the pathogenesis of BA and that polymorphisms of these genes affect susceptibility to BA.     

Effect of Calcium in Assay Medium on D Value of Bacillus stearothermophilus ATCC 7953 Spores

The D value of commercial biological indicator spore strips using Bacillus stearothermophilus ATCC 7953 was increased by higher calcium concentrations in assay media. The calcium concentration in assay media varied among the manufacturers. The calcium concentration in assay media is an important factor to consider to minimize the variation of D value.     

Isolation and Chemical Structure of New Oxidation Product of 5-Ketogluconic Acid,and a Hypothetical Pathway from Glucose to Tartaric Acid through this New Compound

In the course of study on the mechanism of the tartaric acid formation from 5-ketogluconic acid, a new intermediary substance with mauve color to Abdel-Akhel and Smith’s reagent was isolated from intact cell culture liquid. The chemical structure of this substance was determined as 1,2-dihydroxyethyl hydrogen L(+) tartrate from the results of hydrolysis experiments and from the identifications of the constituents of the molecule, and named “pretaric acid.” Tartaric acid was evidently produced from pretaric acid by intact cell culture. Clearly, then, pretaric acid appears to be an intermediate in the formation of tartaric acid from 5-ketogluconic acid. The authors assumed that in the formation of pretaric acid from 5-ketogluconic acid, a Baeyer-Villiger type oxidation occurred.